Compounds and methods for increasing hair growth

ABSTRACT

A method of promoting hair growth can include: a polypeptide having a sequence that has at least 75% complementarity to or at least 75% identical to SPR4; and topically administering the polypeptide to a subject. This can include putting or causing the polypeptide to be in the skin, such as in any dermal layer. In one aspect, the method can include administering the composition topically so as to administer the polypeptide to the subject. In one aspect, the method can include administering the polypeptide to skin of the subject. In one aspect, the method can include administering the polypeptide to a hair follicle of the subject. In one aspect, the method can include administering the polypeptide to a bald spot of the subject.

CROSS-REFERENCE

This patent application claims priority to U.S. Provisional ApplicationNo. 61/930,749 filed Jan. 23, 2014, which provisional application isincorporated herein by specific reference in its entirety.

GOVERNMENT RIGHTS

This technology was made with government support under Grant No.R01AR051598-03 awarded by the National Institute of Health (NIH) andNational Institute of Arthritis and Musculoskeletal and Skin Diseases(NIAMS), and 5R01AR051598-05 and R01 CA173292 awarded by the NationalInstitute of Health (NIH). The government has certain rights in thetechnology.

BACKGROUND

In our recent studies we described a bio-engineered, 4.2 kDa syntheticPHEX-peptide (i.e., SPR4) also referred to as murikal that specificallybinds and neutralizes ASARM-peptides. The SPR4 peptide also corrects themineralization defect in vitro and in vivo and has positive effects onbone regulatory markers. These discoveries (ASARM and SPR4-peptides)have helped provide new strategies to treat select hypophosphatemicbone-mineralization disorders (HYP, ADHR, ARHR, osteoporosis and TIO)and manage hyperphosphatemia in CKD-MBD, ESRD. SPR4-peptide alsoimproves and corrects energy metabolism in healthy mice and mice withhypophosphatemic bone-mineral loss disorders (HYP mice) respectively.The improvements in energy metabolism may have therapeutic utility forosteoporosis, obesity, metabolic syndrome and diabetes. Also,SPR4-peptide in conjunction with a replete phosphate diet may be used totreat inherited hypophosphatemic bone-mineral loss disorders (X-linkedrickets (HYP) and autosomal forms of rickets (recessive and dominant)).

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and following information as well as other features ofthis disclosure will become more fully apparent from the followingdescription and appended claims, taken in conjunction with theaccompanying drawings. Understanding that these drawings depict onlyseveral embodiments in accordance with the disclosure and are,therefore, not to be considered limiting of its scope, the disclosurewill be described with additional specificity and detail through use ofthe accompanying drawings, in which:

FIG. 1 includes data that shows suppression of circulating sclerostin inwild type mice infused with SPR4 using osmotic pump infusion.

FIG. 2 include data that shows suppression of sclerostin in wild typemice cortical bone (femurs) as measured using immunohistochemistry (IHC)following infusion of SPR4 (SPR4-peptide). SPR4 and vehicle were infusedusing osmotic pumps as discussed.

FIG. 3 shows increased active β-catenin protein-expression in wild typemice cortical bone (femurs) as measured by western-blotting followinginfusion of SPR4. SPR4 and vehicle.

FIG. 4 shows murine dermal penetration of fluorescently labeled SPR4 asmeasured using laser confocal microscopy.

FIG. 5 shows accelerated hair growth in depilated mice (N=6) treatedwith repeat localized intradermal (i.d) injections of SPR4 over 11 days.

FIG. 6 shows accelerated hair growth in anagen induced depilated micetopically treated with daily liposome formulations of SPR4 (50 μL).

FIG. 7 shows data for contrast-images from FIG. 6 were digitallyquantitated using a pixel conversion program GelQuant.Net(BiochemicalLabSolutions.com).

FIGS. 8A-8B includes DEXA analysis pictures of representative Vehicleand 5α-DHT male castrated mice showing increased bone and lean mass withtreated mice.

FIG. 9 shows castrated male mice infused with 5α-DHT show increased bonemass, mineral content, lean mass and lean/fat mass ratios compared tovehicle infused castrated male mice (n=6).

FIG. 10 shows high resolution (6 uM) 3D μCT images of femurs fromvehicle and 5α-DHT treated castrated male mice. The arrows highlight themassive increase in cancellous and trabecular bone in the epiphyses ofexperimental mice.

FIG. 11 includes representative pictures of male castrated mice treatedwith vehicle or 5α-DHT.

FIG. 12 shows accelerated hair loss occurred with male mice treated with5α-DHT with an inflection point after 2 months.

FIG. 13 shows the pattern hair baldness is induced in castrated malemice treated with 5α-DHT.

FIGS. 14A-14D show data from intensity dynamic light scattering sizemeasurement and zeta potential of a 15× dilution of example formulations

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein.

Generally, the present technology includes a pharmaceutical preparationthat can increase hair growth when topically applied to the skin. Morespecifically, this technology includes a 4.2 kDa peptide (i.e., SPR4) ina lipid or polymer-based formulation configured for transdermal deliverythat dramatically increases hair growth when topically applied to skin.Also, the present technology relates to use of the novel peptide (i.e.,SPR4) that is bioengineered and modeled from the catalytic binding siteof “Phosphate Regulating Gene with Endopeptidase Homologies” (PHEX) andthe associated molecular pathways that stimulate accelerated hairgrowth.

The SPR4 can increase hair growth when applied to skin. Proof ofincreased hair growth was observed in mice, and it is expected that suchincreased hair growth will occur in all mammals, and be useful forhumans and other mammals to have increased hair growth.

The peptide can be used to reduce or treat or prevent baldness, such aspattern baldness, male pattern baldness, or other types of baldness. Assuch, the peptide can be applied to skin, such as bald skin or skinunder hair. The peptide can be applied to skin that has hair in humansthat do not want to lose their hair, and thereby the peptide can be usedfor hair maintenance. As such, the peptide can be used to increase theduration of having hair and inhibiting the onset of baldness. Thebaldness to be improved by hair growth induced by the peptide can benatural baldness or induced baldness, such as chemotherapy, radiation,or other that causes baldness. The peptide can increase and induce newhair follicle growth, such as in bald spots or regions that have thinhair or even regions that have thick hair. The peptide can be appliedtopically alone or in a pharmaceutical composition. The composition canbe configured for transdermal delivery of the peptide, and thereby caninclude penetration or permeation enhancers, which are well known in thepharmaceutical arts. The composition having the peptide can be appliedto a bald spot or other skin location in a lotion or gel and allowed toabsorb into the skin. Also, the composition can be a shampoo that isapplied to a bald head and/or hair so that the bald spots or thin hairareas or other skin areas can have enhanced hair growth, where theshampoo can be allowed to set for some period of time to allowtransdermal delivery.

In one aspect, the delivery of the peptide may not be true transdermaldelivery because the peptide only needs to enter into the skin forfunctionality. As such, the delivery may be considered to be dermaldelivery or topical delivery or skin delivery; however, transdermaldelivery is allowable. However, transdermal delivery agents, such aspermeation or penetration enhancers that enhance entry of the peptideinto skin can be used as a carrier for the peptide.

The peptide sequence of SPR4 is: NH2TVNAFYSASTNYPRSLSYGAIGVIVGHEFTHGFDNNGRGENIADNG-OH (SEQ ID NO: 1). Thispeptide may be used as shown, or it may be linked on the C-terminal endand/or N-terminal end to other peptides or chemical moieties. Peptidepurity was greater than 90% via HPLC, ion-exchange and also massspectrometry.

In one aspect, the peptide can be applied in a combination cancertherapy and baldness therapy. This can inhibit cancer and promote hairgrowth at the same time. This may be helpful for some skin cancers onbald spots, where the cancer and baldness can be simultaneously treated.For example, a cancer patient with hair loss can be administered thepeptide for treating the cancer and to increase hair growth, such aswhen chemo or radiation has resulted in hair loss.

The peptide can be delivered by any mechanism or system that places thepeptide in the skin and/or follicle and/or near the skin and/orfollicle. The delivery can be by injection into the skin or across theskin. The injection can be a bolus or continuous or sustained.

The delivery can be by topical application. The administration can beone time or repeated in an administration regimen. The administrationcan be topical or dermal or transdermal. The administration can be localor localized. The administration can be by a depot of degradablematerial (e.g., polymer) having the peptide so that degradation releasesthe peptide. The administration may also be systemic, where oral or i.v.administration may be possible.

SPR4 and related peptides directly regulate the Wnt/β-catenin canonicalpathway and several genes important for hair growth. Specifically thecurrent technology relates to the discovery that SPR4 can topicallyinduce and accelerate hair growth and maintain an extended anagen phasewhen applied to the skin in a suitable formulation in the presence oflow concentrations of ZnCl₂. Expression and stability of β-catenin isinfluenced by binding of Wnt to its cell-surface receptors (e.g.,Frizzled or LRP5/6). The binding of Wnt to its co-receptors suppressesGSK kinase, an enzyme that when active phosphorylates active β-catenin(PO4-β-catenin). Phosphorylated β-catenin-PO4 is then targeted forubiquitinylation and proteosomal degradation. Active un-phosphorylatedβ-catenin travels to the nucleus where it binds to specific promotersand transcriptionally regulates target genes. In cells deplete ofβ-catenin due to reduced Wnt signaling, the transcriptional regulationof target genes is switched off. These target genes include thoseimportant for the anagen-phase of hair growth. Of relevance to thecurrent technology, individuals with adrogenetic alopecia (or malepattern baldness), have increased levels of 5-α-reductase (5-αR). Thisenzyme (5-αR) converts testosterone to dehydro-testosterone (DHT) andDHT binds with specificity to the androgen receptor (AR). The DHTandrogen-receptor complex (DHT-AR) then binds with high affinity andspecificity to active β-catenin, and this has the effect of depletingendogenous levels of this important transcriptional regulator(β-catenin). This in turn results in a reduced anagen phase of the haircycle and consequential hair thinning in those males and females withhigh levels of 5-αR. Application of SPR4 suppresses sclerostinexpression and production resulting in increased Wnt signaling andincreased active β-catenin. The increased β-catenin is then able tocompensate for the DHT-AR mediated sequestration of β-catenin moleculesand this restores the transcriptional activation of hair genes. This inturn induces hair growth and lengthens the anagen phase that then curesthe baldness in afflicted individuals. Thus, the polypeptide canformulated for topical administration on the skin to maintain and/orpromote hair growth, such as in a lipid emulsion, liposome or polymerparticle for absorption into the skin and follicles.

SPR4 can mediate hair growth induction by positively affecting hairpattern baldness genes. Specifically, the effects of SPR4 can mediatecorrection of androgen dysregulation of the Wnt-β-catenin signalingpathway in dermal papilla cells from an androgenic alopecia (AGA) ormale pattern baldness scalp. It was found that Wnt signals derived fromundifferentiated matrix cells that give rise to the hair shaft precursorcells activate the Frizzled receptor (Frz) and LPRP5/6 co-receptorcomplex that in turn inactivates glycogen synthase kinase 3β (GSK-3β).When activated, GSK-3β kinase phosphorylates β-catenin (β-catenin-PO4)and this targets the molecule for ubiquitinylation and targetedproteosomal degradation. Wnt mediated inactivation of GSK-3β results inaccumulation of active β-catenin that then translocates to the nucleusand cooperates with Tcf/Lef transcription factors to induce geneexpression required for hair growth. However, in AGA testosterone (T) isefficiently converted to dehydrotestosterone (DHT) due to elevatedlevels of 5α-reductase, resulting in high levels of DHT andandrogen-receptor (AR) that favor AR-β-catenin interactions. TheAR-β-catenin interactions block β-catenin mediated gene expression. SPR4peptide circumvents this process by suppressing sclerostin expression(e. g., a negative regulator of active β-catenin production).

Sclerostin (SOST) suppresses active β-catenin levels by preventingLRP5/6 binding to Frizzled on the plasma membrane. The SOST inhibitionof LRP5/6 binding to Frizzled in turn results in up regulation of GSK30kinase that then phosphorylates β-catenin resulting in targetedubiquitinylation and proteosomal degradation. Thus, the SPR4 mediatedsuppression of SOST and consequential increased β-catenin levelsdynamically compensates for the increased DHT-AR receptor complexsequestration of free β-catenin. This in turn restores genetranscription and normal hair growth in individuals with hair patternbaldness. FIGS. 1, 2 and 3 show changes that lead to suppression ofcirculating sclerostin and increased active β-catenin in wild type miceinfused with SPR4 using osmotic pump infusion.

The positive effects of systemic application of SPR4 on bone aremediated in part by suppression of Sclerostin (SOST), an inhibitor ofthe Wnt β-catenin canonical pathway. Specifically, SPR4 mediatedinhibition of sclerostin induces active β-catenin that in turn has ananabolic effect on bone.

Of relevance to the present technology the Wnt/b-catenin canonicalpathway plays a major role in regulating hair growth. Specifically, thepresent technology is directed to compositions and methods for promotinghair growth in individuals with androgenic alopecia (AGA) and also otherhair baldness conditions by targeting a specific event in theWnt/β-catenin canonical-pathway. In particular, a novel SOST-inhibitorpeptide SPR4 and related compounds are provided. Local application ofSPR4 and related peptides to the scalp and skin accelerates and sustainshair growth by lengthening the anagen phase and inducing β-cateninexpression.

The SPR4 peptide and topical formulation overcomes delivery andadministration and regimen issues. Previously, a primary issue with theformulation and subsequent treatment using non-small molecule drugs,such as peptides and macromolecules, has been the very poor biologicalabsorption by routes other than intravenous, subcutaneous, andintramuscular. Previous treatments that may be self-administered by thepatient on a periodic basis, such as several times a week, daily or morefrequently, are inconvenient and difficult to administer by theseroutes. In addition, most parenteral routes result in systemicabsorption and distribution, which can result in both sub-therapeuticdrug levels at the intended site (e.g. the follicles or the dermis for ahair growth enhancing product) and non-specific drug actions in othertissues. Thus, the topical compositions with SPR4 offer an advantage.

Topical delivery is the preferred method of treatment with a SPR4peptide hair growth enhancer since these formulations are convenient forthe patient to apply and application can be limited to the areas whereenhanced hair is desired. Thus, this technology is a non-irritatingformulation that provides delivery of the polypeptide into the hairfollicles and the dermis. Also, the formulation controls absorption intothe dermis to limit the potential for systemic absorption via the lymphand hematological system, which can lead to dilution of the drug in theintended treatment area and the potential for exposing non-intendedtissues to the drug.

The SPR4 hair enhancing products can be typically applied periodically,from several times a week, daily, to several times a day, and theapplication is often to public areas of the body, hence the formulationshould be non-irritating and not cause discoloration of the skin. Lipidand polymeric formulations as described in this technology can delivertherapeutic levels of the SPR4 peptide into the hair follicles, whilelimiting dermal absorption, and not causing significant irritation ordiscoloration of the skin.

Peptide loaded liposomes were created using L-α-phosphatidylcholine (10w/v %), ethanol (4.3 v/v %) and SPR4 (0.099% w/v). The ethanolic lipidmixture was vortexed for 5 minutes and sonicated until fully dissolved.SPR4 in PBS was then added while stirring. PBS was then added drop wisewhile stirring until the final concentration was reached. Loading degreeof SPR4 within the liposome was determined using EZStart 7.4 softwareand a Shimadzu 2010CHT system with a TSKgel ODB 10 oz column (4.6 mm IDX 25 cm, 5 μm) using ddH2O w/0.002% TFA and ACN w/0.002% TFA (1 ml/min).Organic solvent concentration within the method; 10% 0-5 minutes, linearincrease to 90% 5-10 minutes, 90% 10-15 minutes, linear decrease to 10%15-15.1 minutes and 10% 15.1-20 minutes. Retention time of the SPR4peptide was found to be 10.35 minutes. Loading degree of most recentformulation method found to be 41.35% (w/w).

The loaded liposomes can be included in a micro or nano emulsion. Theliposomes can be considered to be a pharmaceutical carrier. The micro ornano emulsions can be considered to be pharmaceutical carriers for theliposomes.

In one embodiment, a topical composition can include: a pharmaceuticalcarrier configured for topical application to a subject; and apolypeptide in the pharmaceutical carrier and having a sequence that hasat least 75% complementarity to or at least 75% identical to SPR4,wherein SPR4 is: TVNAFYSASTNYPRSLSYGAIGVIVGHEFTHGFDNNGRGENIADNG (SEQ IDNO: 1). In one aspect, the polypeptide has at least 80% complementarityto or is at least 80% identical to SPR4. In one aspect, the polypeptidehas at least 90% complementarity to or is at least 90% identical toSPR4. In one aspect, the polypeptide has at least 95% complementarity toor is at least 95% identical to SPR4. In one aspect, the polypeptide hasat least 99% complementarity to or is at least 99% identical to SPR4. Inone aspect, the polypeptide has 100% complementarity to or is 100%identical to SPR4.

In one embodiment, the polypeptide is included in a fusion polypeptidewith second polypeptide. The second polypeptide can be any polypeptide,which can be on the N- or C-terminus. The polypeptide can providebeneficial properties, such as water solubility, receptor targeting,endosomal escape, hair growth promotion, cell growth promotion, or anyother benefit. In one aspect, the endosomal disrupting polypeptideincludes PC4 or derivative thereof.

In one embodiment, the polypeptide is present in an amount sufficient toincrease hair growth in a subject upon topical application to thesubject. In one aspect, the polypeptide is present in an amountsufficient to increase hair follicle growth/development in a subjectupon topical application to the subject.

In one embodiment, the polypeptide is dissolved in the carrier. In oneaspect, the carrier includes one or more of cetearyl alcohol, cetearylglucoside, squalane, isopropyl palmate, octyldodecaonol, phenoxyethanol,methylparaben, etheylparaben, butylparaben, propylparaben,isobutylparaben, glycerin, butylene glycol, sodium acrylate,acryloyldimethyl taurate, isohexadecane, polysorbate, glyceryl stearate,dicaprylyl ether, alkyl benzoate, isononyl isononanoate,methylpropanediol, tetrasodium EDTA, iodoproynyl butylcarbamate,triethanolamine, ketoconazole, serenoa serrulata extract, emu oil,niacin vitamin B3, caffeine, pyridoxine, L-pathenol, linolenic acid,simmondsia chinesis seed oil, zinc oxide, lecithin, ZnCl₂,L-α-phosphatidylcholine, ethanol, PBS, phospholipids, fatty acids,tocopherol, and derivatives thereof and equivalents thereof. In oneaspect, the polypeptide is contained in a liposome or microsphere orpolymer particle or lipid emulsion or combination thereof, and such canbe included in a carrier.

In one embodiment, the composition can also include an active hairgrowth agent. In one aspect, the active hair growth agent is selectedfrom minoxidil and finasteride.

In one embodiment, the composition promotes an anagen hair growth phase.In one embodiment, the composition promotes a catagen hair growth phase.In one embodiment, the composition promotes a telogen hair growth phase.In one embodiment, it can be any one or more or all three of these hairgrowth phases.

In one embodiment, the composition treats alopecia and related symptomsand related methods thereof. This can include administering a sufficientamount for any or the methods or treatments or hair growth maintenanceas described herein. People with full thick hair can use in methods tomaintain the full thick hair.

A method of promoting hair growth can include: a polypeptide having asequence that has at least 75% complementarity to or at least 75%identical to SPR4; and topically administering the polypeptide to asubject. This can include putting or causing the polypeptide to be inthe skin, such as in any dermal layer. In one aspect, the method caninclude administering the composition topically so as to administer thepolypeptide to the subject. In one aspect, the method can includeadministering the polypeptide to skin of the subject. In one aspect, themethod can include administering the polypeptide to a hair follicle ofthe subject. In one aspect, the method can include administering thepolypeptide to a bald spot of the subject. In one aspect, the method caninclude administering the polypeptide so as to modulate theWnt/beta-catenin canonical pathway.

In one aspect, the method can include administering the polypeptide toregulate one or more genes involved in hair growth. In one aspect, themethod can include administering the polypeptide so as to acceleratehair growth. In one aspect, the method can include administering thepolypeptide so as to lengthen the anagen phase. In one aspect, themethod can include administering the polypeptide so as to reduce5-alpha-reductase. In one aspect, the method can include administeringthe polypeptide so as to suppress sclerostin. In one aspect, the methodcan include administering the polypeptide so as to increase Wntsignaling. In one aspect, the method can include administering thepolypeptide so as to increase active beta-catenin. In one aspect, themethod can include administering the polypeptide in an amount to treatalopecia and/or related syndromes. In one aspect, the method can includeadministering the polypeptide in an amount to lengthen the anagen phase.In one aspect, the method can include administering the polypeptide inan amount to increase hair growth on a bald spot. In one aspect, themethod can include administering the polypeptide in an amount toincrease hair follicle growth. In one aspect, the method can includeadministering the polypeptide in an amount to increase hair growthcompared to when the polypeptide is not administered.

Accordingly, castrated B6CBAF1/J hybrid male mice treated with 5-αDHTinfused pellets provide for the first time a powerful and new model forthe study of AGA in mice.

In one aspect, the SPR4 peptide can be formulated into a gel. The gelmay or may not include the liposome or other particle having the SPR4encapsulated therein.

In one aspect, delivery of the SPR4 peptide to skin can be viamicroneedle application. A derma-roller application system that hasmicroneedle disk rollers (high quality medical grade steel) can deliverthe SPR4 peptide. This system is used in patients for the dermalapplication (cosmetic and medical) of medications and cosmeticpharmaceuticals in humans. The system is highly effective, painless andnoninvasive (no blood released).

The micro-roller provides a direct way of introducing SPR4 through thedermis into the circulation or into the sub-dermis and can be safelyused on the face or scalp. The skin is not visibly damaged and themicro-pores opened by the system close with one hour.

The technology also relates to variant forms of these sequences and/orof these fragments. The expression “variant” indicates a polypeptide ora peptide that differs, for example, from the sequence of a referencepeptide while keeping its essential properties. Generally, thedifferences are limited so that the sequences of the reference peptideand those of the variant are quite similar and, in some regions,identical.

Preferentially, the variant forms are those which vary from referencesequences by the substitution of chemically equivalent (or homologous)amino acids, that is, by the substitution of a residue with anotherpossessing the same characteristics. Thus, classical substitutions takeplace between Ala, Val, Leu and Ile; between Ser and Thr; between theacid residues Asp and Gln; and between the basic residues Lys and Arg,or between the aromatic residues Phe and Tyr.

The expression “variant” indicates a polypeptide or a peptide thatdiffers, for example, from the sequence of a reference peptide whilekeeping its essential properties. Generally, the differences are limitedso that the sequences of the reference peptide and those of the variantare quite similar and, in some regions, identical. A variant peptide anda reference peptide may differ in their amino acid sequence by one orseveral substitutions, additions, or deletions in all the combinations.

In the technology, the term “amino acid” refers to any natural orunnatural organic acid having the formula (II): —NHR—CR—C(O)—O (II),where each —R is independently selected from a hydrogen or an alkylgroup having between 1 and 12 carbon atoms. Preferentially, at least an—R group of each amino acid is a hydrogen. The term “alkyl” refers to acarbon chain that can be linear or branched, substituted (mono- orpoly-) or not substituted; saturated, mono-saturated (a double or triplebond in the chain), or poly-unsaturated (two or several double bonds,two or several triple bonds, one or several double bonds, and one orseveral triple bonds in the chain).

Many biologically compatible forms of protection can be considered, suchas acylation or acetylation of the amino terminal end, or amidation oresterification of the terminal carboxyl end. Such forms are well knownby those skilled in the art. Thus, the technology relates to the use aspreviously defined and is characterized by the fact that the peptideeither is or is not in a protected form. Preferably, the protection usedis either acylation or acetylation of the amino terminal group, oresterification or amidation of the terminal carboxyl end, or both ofthem. The amino acid derivatives and the peptide derivatives also relateto amino acids and peptides bound together by a pseudo-peptide bond. Bythe term “pseudo-peptide bond,” we refer to all types of bonds likely toreplace “classical” peptide bonds.

In the domain of amino acids, the geometry of the molecules is such thatthey can be theoretically presented as different optical isomers. Thereis indeed a molecular conformation of the amino acid (AA) such that itdeviates to the right of the plane of polarization of the light(dextrorotatory conformation or D-aa), and a molecular conformation ofthe amino acid (aa) such that it deviates to the left of the plane ofpolarization of the light (levorotatory conformation or L-aa). Natureretained for the natural amino acids only levorotatory conformation.Consequently, a peptide of natural origin will be made up only of aminoacids of type L-aa. However, chemical synthesis in a laboratory makes itpossible to prepare amino acids having two possible conformations.

From this basic material, it is thus possible to incorporate, duringpeptide synthesis, amino acids in the form of dextrorotatory orlevorotatory optical isomers. Thus, the amino acids constituting thepeptide according to the technology, can be under configuration L- andD-; in a preferential way, amino acids are in L configuration. Thepeptide according to the technology can be in L, D, or DL-configuration.

According to the technology, the peptides can be prepared using allappropriate methods. Thus, the peptides can be isolated peptides frompeptides and proteins existing naturally, recombinant peptides,synthetic peptides, or peptides produced by a combination of thesemethods. Of course, the methods, in order to prepare the peptidesaccording to the technology, are well known by one skilled in the art.Thus, the peptide according to the technology may be of natural orsynthetic origin. Preferentially, according to the technology, thepeptide is obtained by chemical synthesis.

According to an advantageous mode of embodiment of the technology, theabovementioned peptides are solubilized beforehand in one or severalcosmetically or acceptable solvents classically used by one skilled inthe art, such as water, ethanol, propylene glycol, butylene glycol,dipropylene glycol, ethoxylated or propoxylated diglycols, cyclicpolyols, vaseline, a vegetal oil, or any combinations of these solvents.

According to another advantageous mode of embodiment of the technology,the abovementioned peptides are solubilized beforehand in one cosmeticor vector such as liposomes, emulsions, solid lipid nanoparticles,micelles, micro- or nano-particles or adsorbed on powdery organicpolymers, mineral supports like talcs and bentonites, and more generallysolubilized in, or fixed on, any cosmetically or acceptable vector. Thevector may be a particle with a range of sizes between 10 nm and 100microns, more specifically 20 microns and 20 nm, and more specifically15 microns and 100 nm.

Particles may be composed of one or several excipients, includingexcipients generally regarded as safe (GRAS) by the FDA; phospholipids;saturated and unsaturated fatty acids and esters; PEGs of sizes from 100g/mol to 20,000 g/mol, more specifically PEGs of 200 to 1200 g/mol, andmore specifically PEGS of 400 to 600 g/mol; natural and unnaturalpolyamino acids having hydrophilic, hydrophobic, or chemically modifiedresidues or some combination of these; synthetic and semisyntheticpolymers such as HPMA, poly-lactic acid, and poly-esters; cyclodextrins;alcohols having one to 20 carbons; quaternary amines; lipids; fats;hydrophilic polymers; hydrophobic polymers; hydrogels; proteins; and anycombination of the above or derivatives of the above.

Further, particles may be engineered for uptake, delivery, or entrapmentinto the follicles, and the particles may limit or control the deliveryof peptides or drugs into dermis surrounding the follicles, and theparticles may limit or control the absorption of drugs or peptides intothe blood, lymph, and systemic circulation and non-skin tissues.

It is of course obvious that the peptide according to the technology canbe used alone or in association with at least one other active agent, inor for the preparation of a cosmetic and/or dermatological and/orpharmaceutical composition.

α-Tocopherol, a Vitamin E derivative, is a widely used additive intopical medications and cosmetics for its natural aesthetics. However,isolated cases have shown tocopherol may induce allergic contactdermatitis when frequently applied topically. Topical immunotherapy is acommon method of treatment for acute cases of alopecia areata. Thetopical sensitizer is applied in increasing amounts to the inflictedskin, commonly the scalp, until an eczematous response is observedindicating sensitization to the material.

Multiple theories on the mechanism of action of topical immunotherapyexist including the competitive antigenic inhibition of the auto immuneresponse associated with alopecia areata upon allergic suppresser T cellgeneration. For this reason, addition of α-tocopherol to a formulationdirected at treating alopecia related ailments could be beneficial. Inone aspect, a micro or nano emulsion can include the tocopherol toenhance delivery of the peptide. Also, compositions having the peptideloaded into a particle can be formulated with the tocopherol.

In one embodiment, a particle having the peptide can be a certain sizeor within a certain size distribution. FIG. 14 show particle size data.The size and zeta potential can be important as they are factors thatimpact the absorption of the formulation and peptide topically. Theformulation actually passes through the follicle and could be moreproperly called transappendageal drug delivery. Thus, one embodiment istransappendageal delivery and not transdermal delivery.

In one embodiment, the combination of a nano emulsion with the SPR4peptide can allow for target delivery into the perifolicular region atthe right concentrations where the peptide can have a therapeuticeffect. Such delivery can avoid negative effects in a healthy individualthat may occur if the peptide is given systemically (e.g., inhibitsproblems related to bone growth). Accordingly, localization and thecorrect peptide levels in those tissues may be important for asuccessful therapy. The emulsion may or may not have the peptideencapsulated in a particle.

If SPR4 is administered continuously (e.g., infusion) then there may benegative effects on bone. As such, one aspect of the invention is devoidof continuous infusion of the peptide. If administered intermittently(e.g., bolus) then the peptide has an anabolic or positive effecton-mineral metabolism and also glucose tolerance and insulinsensitivity. As such, one aspect of the invention allows for bolusadministration. The SPR4-peptide does not cause osteosarcoma in mice.

EXAMPLES

SPR4-peptide was synthesized by Polypeptide Laboratories (San DiegoCalif., USA 92126) with purity over 90%. Two batches of peptide weresynthesized as follows: (1) Biotinylated (50 mg); and (2) nonbiotinylated (950 mg). The biotinylated form was to be used to assessdermal penetration of peptide following topical application usingconfocal microscopy. A novel model using a murine strain (B6CBAF1/Jhybrid mouse) was developed and implemented.

L-α-Phosphatidylcholine (2.39 g/mL) was dissolved in ethanol. Thesolution was vortexed for 5 minutes and sonicated for 10 minutes oruntil a clear solution was achieved. Subsequently, SPR4 (5 mg/mL) wasdissolved in PBS, pH 7.4, vortexed for 2 minutes and sonicated for 10minutes or until dissolved. The SPR4 solution was added dropwise (1.5mL/h) to the lipid ethanolic solution using syringe pump whileconstantly stirring. Then additional PBS (3.8× to SPR4 solution volume)was added dropwise (1.5 mL/h) using syringe pump while constantlystirring. The final solution was stored at 4° C. and covered from lightexposure. The final formulation contained 103.8 mg/mL of lipid, 4.3%(v/v) of ethanol, and 0.99 mg/mL of SPR4.

L-α-Phosphatidylcholine/Cholesterol/1,2-dioleoyl-3-trimethylammonium-propane(DOTAP)/α-Tocopherol were dissolved in chloroform:methanol (2:1 (v/v))to 8.35 mg/ml with a molar ratio of 55.7:34.3:8.6:1.3, PC:CH:DOTAP:α-Tocopherol. The solution was vortexed for 5 minutes and sonicated for10 minutes or until a clear solution was achieved. SPR4 (0.165 mg/mL)was dissolved in 25 mM acetic acid with subsequent PBS, pH 7.4 addition(1:9, AcOH:PBS). The SPR4 solution was added dropwise (1.5 mL/h) to thelipid solution using syringe pump while constantly stirring until a 6:1solvent-to-buffer ratio was achieved. The solution was then sonicatedfor 2-5 minutes or until clear. If mixture was not clear aftersonication, methanol was added to no more than 10% of the total volume.Subsequently, organic solvents were removed by rotoevaporation. Solventremoval continued until all foaming within the mixture ceased.

The final solution was stored at 4° C. and covered from light exposure.The final formulation contained 50 mg/mL of lipid and 0.99 mg/mL ofSPR4.

L-α-Phosphatidylcholine/Cholesterol/1,2-dioleoyl-3-trimethylammonium-propane(DOTAP)/α-Tocopherol were dissolved in ethanol to 125 mg/ml with a molarratio of 55.7:34.3:8.6:1.3, PC:CH:DOTAP: α-Tocopherol. The solution wasvortexed for 5 minutes and sonicated for 10 minutes or until a clearsolution was achieved. SPR4 (1.73 mg/mL) was dissolved in 25 mM aceticacid with subsequent PBS, pH 7.4 addition (1:9, AcOH:PBS). The lipidethanolic mixture was heated to 65° C. for 2-3 min and the SPR4 solutionheated to 37° C. for 2-3 min. The lipid mixture was added dropwise tothe SPR4 solution while constantly stirring until a 2:3solvent-to-buffer ratio was achieved.

Subsequently, the solution was passed ten times through a 100 nmpolycarbonate filter and dialyzed against PBS for 24 hours to removeexcess ethanol. Ethanol was added to 4.3% (v/v) following dialysis. Thefinal solution was stored at 4° C. and covered from light exposure. Thefinal formulation contained 50 mg/mL of lipid, 4.3% (v/v) of ethanol and0.99 mg/mL of SPR4.

Glyceryl dilaurate/Cholesterol/Polyoxyethylene (10) stearyl ether(POE-10)/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/α-Tocopherolwere mixed with a weight ratio of 50:14:23:12:1,GDL:CH:POE-10:DOTAP:α-Tocopherol and melted at 70° C. The lipid melt wasthen filtered through a 0.22 μm filter and reheated to 70° C. prior tobeing drawn into a sterile syringe. A second syringe containing PBS washeated to 65° C. and attached to a three way stopcock with the lipidmixture syringe. The aqueous phase was slowly injected into the lipidphase syringe. Subsequently, the mixture was rapidly passed back andforth between the two syringes while being cooled under water untilreaching room temperature. The mixture was then sonicated for 20 min.SPR4 (2.0 mg/mL) was dissolved in 25 mM acetic acid with subsequent PBS,pH 7.4 addition (1:9, AcOH:PBS). Equal volumes of the empty liposomesolution and SPR4 solution were mixed and incubated at room temperaturefor 45 min. The final solution was stored at 4° C. and covered fromlight exposure. The final formulation contained 50 mg/mL of lipid and 1mg/mL of SPR4.

Glyceryl dilaurate/Cholesterol/Polyoxyethylene (10) stearyl ether(POE-10)/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/α-Tocopherolwere mixed with a weight ratio of 50:14:23:12:1,GDL:CH:POE-10:DOTAP:α-Tocopherol and melted at 70° C. The lipid melt wasthen filtered through a 0.22 μm filter and reheated to 70° C. prior tobeing drawn into a sterile syringe. A second syringe containing PBS washeated to 65° C. and attached to a three way stopcock with the lipidmixture syringe. The aqueous phase was slowly injected into the lipidphase syringe. Subsequently, the mixture was rapidly passed back andforth between the two syringes while being cooled under water untilreaching room temperature. The mixture was then sonicated for 20 min.SPR4 (2.0 mg/mL) was dissolved in 25 mM acetic acid with subsequent PBS,pH 7.4 addition (1:9, AcOH:PBS). Equal volumes of the empty liposomesolution and SPR4 solution were mixed and incubated at room temperaturefor 45 min. Ethanol was added to 4.3% (v/v). The final solution wasstored at 4° C. and covered from light exposure. The final formulationcontained 50 mg/mL of lipid, 4.3% (v/v) of ethanol and 1 mg/mL of SPR4.

Glyceryl dilaurate/Cholesterol/Polyoxyethylene (10) stearyl ether(POE-10)/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/α-Tocopherolwere mixed with a weight ratio of 50:14:23:12:1,GDL:CH:POE-10:DOTAP:α-Tocopherol and dissolved in ethanol to (33 mg/ml).The solution was vortexed for 5 minutes and sonicated for 10 minutes oruntil a clear solution was achieved. Subsequently, SPR4 (1 mg/mL) wasdissolved in PBS, pH 7.4, vortexed for 2 minutes and sonicated for 10minutes or until dissolved. The SPR4 solution was added dropwise (1.5mL/h) to the lipid ethanolic solution using syringe pump whileconstantly stirring. Subsequently, ethanol was removed byrotoevaporation. The final solution was stored at 4° C. and covered fromlight exposure. The final formulation contained 50 mg/mL of lipid, 0%(v/v) of ethanol, and 0.99 mg/mL of SPR4.

Glyceryl dilaurate/Cholesterol/Polyoxyethylene (10) stearyl ether(POE-10)/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/α-Tocopherolwere mixed with a weight ratio of 50:14:23:12:1,GDL:CH:POE-10:DOTAP:α-Tocopherol and dissolved in ethanol to (33 mg/ml).The solution was vortexed for 5 minutes and sonicated for 10 minutes oruntil a clear solution was achieved. Subsequently, SPR4 (1 mg/mL) wasdissolved in PBS, pH 7.4, vortexed for 2 minutes and sonicated for 10minutes or until dissolved. The SPR4 solution was added dropwise (1.5mL/h) to the lipid ethanolic solution using syringe pump whileconstantly stirring. Subsequently, ethanol was removed byrotoevaporation. Ethanol was added to 4.3% (v/v). The final solution wasstored at 4° C. and covered from light exposure. The final formulationcontained 50 mg/mL of lipid, 0% (v/v) of ethanol, and 0.99 mg/mL ofSPR4.

The encapsulation efficiency of the SPR4 into a liposome was determinedas stated below. A known volume of the final liposome solution wascentrifuged at 15,000×g for 15 min. The supernatant, unencapsulatedSPR4, was removed and placed in a new centrifuge cuvette and the pellet,encapsulated SPR4, was retained. This procedure was repeated twice oruntil a pellet was no longer visible after centrifugation. Thesupernatant was sufficiently diluted with PBS and the concentration ofSPR4 determined by RP-HPLC against a known standard concentrationprofile. The pellets were combined and dissolved in ethanol and theconcentration of SPR4 determined by RP-HPLC against a known standardconcentration profile. This procedure was repeated for all formulations.

Example for Production of Empty Liposomes.

The liposome vesicle was formulated with the core starting materialL-α-phosphatidylcholine from egg yolk, ethanol as a penetration enhancerand phosphate buffered saline (PBS) to stabilize the vesicle insolution: Measure 377.4 mg Egg-PC (L-α-phosphatidylcholine); Add 157.8uL Ethanol (200 Proof); Vortex mixture (˜5 min) and sonicate mixture(˜10 min) until Egg-PC fully dissolved; Add 3360.0 uL PBS, pH 7.4,dropwise while constantly stirring. PBS dispersed via an auto syringeinstrument at 1.5 mL/h.; and Store at 4° C., covered from lightexposure.

Example of Peptide Loaded Liposomes

The SPR4 peptide was formulated in the vesicles: Measure 377.4 mg Egg-PC(L-α-phosphatidylcholine); Add 157.8 uL Ethanol (200 Proof); Vortexmixture (˜5 min) and sonicate mixture (˜10 min) until Egg-PC fullydissolved; Add 720 uL peptide solution (5 mg/mL) dropwise whilestirring; Peptide stock (5 mg/mL) solution; Measure 4.06 mg driedpeptide; Add 812 uL PBS (ph 7.4); Vortex for 2 min; Sonicate for −10 minor until peptide fully dissolved; Store at −20° C., covered from lightexposure; Add 2760 uL PBS, pH 7.4, dropwise while constantly stirring.PBS dispersed via auto syringe instrument at 1.5 mL/h; and Store at 4°C., covered from light exposure.

Lysine modified chitosan example: Reference: Biomaterials 31 (2010)4129-4138. First, lysine modified chitosan (LMC) is prepared as follows.0.11 g of chitosan was dissolved in 10 mL dd H2O, followed by additionof 5 mL HCl (3 mol/L). Butyloxycarbonyl modified lysine, EDC, and DMFare consequently added into the solution under stirring.

Polymerization was carried out at 25 C overnight. Then thebutyloxycarbonyl groups are removed by the addition of TEA. The productis centrifuged and washed with ethanol three times. Subsequently,peptide is reacted with the lysine modified chitosan in the presence ofEDC at pH 5. The reaction is carried out with stirring for 24 hours. Theresulting peptide polymer conjugates were dialyzed again water for 48hours with 4 water changes.

Calcium phosphate method: Reference: International Journal ofPharmaceutics 250 (2003) 25. First, sodiumbis(ethylhexyl)sulphosuccinate (SBS, 0.1 M) in hexane solution wasprepared. Ten milliliters of CaCl2 (20% w/v), 80 mL dd H2O and 1 mL ofpeptide solution were dissolved by continuous stirring for 48 h to formsolution A. Second, another SBS in hexane solution was prepared. Tenmilliliters of Na2HPO4 (5% w/v), 70 mL of dd H2O and 1 mL of peptidewere dissolved by continuous stirring for 48 h to form solution B.Third, solution B was slowly added to solution A with continuousstirring for 6 h. The resulting nanoparticles are centrifuged for 30 minat 1000 rpm and washed with hexane three times. Finally it wasredispersed in 5 ml of dd H2O by sonication.

Conjugate peptide to low-molecular-weight protamine: Reference: J PharmSci. 2013 November; 102(11):4109-20. Peptide is conjugated via adegradable linker, such as an ester or hydrazone, to protamine polymer.The protamine may be of a size of x to x. An amide linkage between theprotected peptide can be made using EDAC/NHS chemistry to conjugate theC-terminus an amine of protamine, followed by deprotection. A hydrazonecan be formed by conjugate of an aldehyde group to the N or C terminusof the peptide followed by formation of the Schift base under mild acidconditions (e.g. pH 3-5), followed by purification by a means such asdialysis or solvent precipitation. The ester can be formed using the Cterminus or a side chain (such as aspartic or glutamic acid) and analcohol conjugated to the protamine.

FIG. 1 shows suppression of circulating sclerostin in wild type miceinfused with SPR4 using osmotic pump infusion. Here, male (5-week)C57B/L6 mice were used for the study represented in FIGS. 1 to 3. Allmice were maintained on a 1% phosphorus and 2.4 IU/g Vitamin-D3 diet(Harlan Teklad Rodent Diet 8604, Indianapolis, Ind.). Mice (5 week) weresurgically implanted with Alzet osmotic pumps (Durect Corporation,Cupertino, Calif.) and infused with SPR4-peptide (276 nmoles/hr/kg) orvehicle (0.9% physiological Saline; VE) for 28 days. Specifically, SPR4peptide was dissolved as follows: (1) 200 μg of peptide (SPR4) is firstdissolved in 20 μL of 25 mM acetic acid, (2) 180 μL of 50 mM TrispH7.4/150 mM NaCl is then added, (4) 4 μL of 1 mM ZnCl₂ is added last(note ZnCl₂ can be added after the peptide is dissolved in aqueoussolutions 1 and 2 to prevent precipitation of peptide). The SPR4 peptidewas first completely dissolved in 25 mM acetic acid (20 μg/uL) followedby the addition of 50 mM Tris pH7.4/150 mM NaCl to give a finalconcentration of 2 ug/uL. Finally, ZnCl₂ from a stock 1 mM solution wasadded to give a final concentration of 20 uM ZnCl₂. The procedure wasfound to dissolve peptide and the addition of ZnCl₂ was done lastbecause earlier additions of ZnCl₂ result in precipitation of peptide.Alzet pump model #2004 with a constant infusion rate of 0.25 uL/h over28 days was used. 2 groups were studied (n=6/group); (1) Wild type miceinfused with vehicle (WT-VE), and (2) Wild type mice infused with SPR4peptide (WT-SPR4) At specific intervals throughout the experiment asdetailed in the results section tail blood-samples were collected inserum-separator tubes. On the final day of the infusion experiment (day28) blood and urine were collected from mice fasted overnight inmetabolic cages with full access to water (1 cage/mouse). The blood fromthe final bleed was collected by cardiac exsanguination and serumurinalysis. Briefly, Osteocalcin (Mouse Osteocalcin EIA Kit; BTI,Stoughton, Mass.), alkaline phosphatase (Liquid Alkaline Phosphatase;Pointe Scientific Inc, Canton, Mich.), 1.25 (OH)2D3 (IDS Inc., FountainHills, Ariz.) and FGF23 (Kainos Laboratories Inc., Tokyo, Japan) weremeasured on serum samples. Inorganic phosphorus, calcium, creatinine(Pointe Scientific Inc, Canton, Mich.) and Osteopontin (Quantikine MouseOsteopontin; R&D Systems, Minneapolis, Minn.) levels were assessed bothin serum and urine. A competitive ELISA kit was used for the peptidemeasurement. Circulating sclerostin (SOST) was measured using acommercially available ELISA kit purchased from ALPCO Diagnostics(Keewaydin Drive, Salem, N.H. 03079, USA).

FIG. 2 shows suppression of sclerostin in wild type mice cortical bone(femurs) as measured using immunohistochemistry (IHC) following infusionof SPR4. SPR4 and vehicle were infused using osmotic pumps as discussed.It was found that (A Images) sclerostin protein-expression (brown-stain)in femur bone-sections is localized to osteocytes (see arrows) andmarkedly suppressed in SPR4 treated mice (e.g., compare photos in Panels1 and 2 of A Images). Magnifications are 60× and are from representativecortical femur sections (matched regions). It was found that (B Images)sclerostin protein-expression (brown-stain) in renal cortex sections ismarkedly suppressed in SPR4 treated mice (e.g., compare photos 1 and 2of B Images). Staining is localized to renal tubules with littleglomerular staining. Magnifications are 20× and are from representativesections (matched regions). Immunohistochemistry was carried out.

Specifically, proximal tibias and distal femurs were decalcified in 0.1M EDTA aqueous solution for 2 weeks until complete demineralization.Decalcified bones and left kidneys were dehydrated in absolute ethanoland embedded in paraffin. 5 μm thick sections were cut on a rotarymicrotome. Sections were dried overnight on pre-charged pre-cleanedslides (VWR Scientific, PA, USA), de-paraffinized and rehydrated. Forimmunohistochemistry, after antigen retrieval by incubation in citricacid buffer 10 mM pH 3 for 60 minutes at 37° C., nonspecific sites wereblocked with 1× animal free blocker (Vector Laboratories Inc., CA, USA)and then sections were incubated with specific primary antibodies for 1hour. An Immunohistological Vectastain ABC kit (Vector LaboratoriesInc., CA, USA) is routinely used and slides counterstained with DAPI ormethyl-green, dehydrated and mounted with entellan. Polyclonal primaryantibodies and the in situ hybridization was performed. Probes werelabeled with fluorescein tag using the FastTag Basic Labeling Kit anddetected with an alkaline phosphatase anti-fluorescein (VectorLaboratories Inc., CA, USA) antibody according to the manufacturer'sinstructions.

FIG. 3 show increased active β-catenin protein-expression in wild typemice cortical bone (femurs) as measured by western-blotting followinginfusion of SPR4. SPR4 and vehicle were infused using osmotic pumps asdescribed in connection with FIG. 1. The increased active β-catenin asillustrated by calculating the ratio of active (e.g., nonphosphorylated)and inactive phosphorylated β-catenin is consistent with the suppressedsclerostin shown in FIGS. 1 to 4. Western analysis of protein lysatesprepared from femurs frozen in LN2 were undertaken. Each lane was loadedwith same amount of protein for each sample. Transferrin was used as aninternal control for chemiluminescent pixel density calculations (BioRadQtyl software and FluorS MaX Imaging). One way Anova and Newman-KeulsPost Test was used to calculate statistical significance (P<0.01* andP<0.001**).

Some data shows the decreased expression of sclerostin (SOST) mRNA frombone (femurs) with changes in expression of other genes in mice infusedwith SPR4.

Additional data shows the decreased expression of sclerostin (SOST) mRNAfrom whole kidneys with changes in expression of other genes in miceinfused with SPR4.

FIG. 4 shows murine dermal penetration of fluorescently labeled SPR4 asmeasured using laser confocal microscopy. SPR4 is visualized by thegreen color and nuclear DAPI-staining blue. Rows A, B, C depictdifferent magnifications and dermal layers as indicated in the figure.The left and right pictures of Rows A, B and C are SPR4 and DAPI nuclearstained images respectively and the middle pictures are merged images.

SPR4 peptide (1 mg) was fluorescently labelled using a commerciallyavailable kit purchased from Thermo Scientific (#53024, DyLight™ 488Antibody labeling kit (A_(max) 493 nM); Pierce Biotechnology, Rockford,Ill. 61105 USA). A liposome peptide formulation was prepared asdescribed in connection to FIG. 6 and 50 μL applied to anagen phaseinduced dorsal-hair depilated mice (also FIG. 6). Mice were euthanizedafter 30 min and paraffin sections of 10% neutral buffered formalin (4%formaldehyde in phosphate buffered saline) fixed skin prepared forhistology.

FIG. 5 shows accelerated hair growth in depilated mice (N=6) treatedwith repeat localized intradermal (i.d) injections of SPR4 over 11 days.Specifically, SPR4 peptide was dissolved as follows; 200 μg of peptide(SPR4) is first dissolved in 20 μL of 25 mM acetic acid, 180 μL of 50 mMTris pH 7.4/150 mM NaCl is then added, 4 μL of 1 mM ZnCl₂ is added last(note ZnCl₂ should be added after the peptides is dissolved in aqueoussolutions 1 and 2 to prevent precipitation of peptide). This gives afinal concentration of 0.98 mg/mL (SPR4) in 44 mM Tris pH7.4/132 mMNaCl/19.6 μM ZnCl₂. Single 100 uL i.d injections (98 μg) were give eachday of dissolved SPR4 peptide (experimental) or vehicle (44 mM TrispH7.4/132 mM NaCl/19.6 μM ZnCl₂). Anagen phase in 5 week old mice wildtype mice (C57BL/6) was induced by depilation of their dorsal hair usingWax Strips (Del Laboratories, Farmingdale, N.Y.) following themanufacturer's instructions.

FIG. 6 shows accelerated hair growth in anagen induced depilated micetopically treated with daily liposome formulations of SPR4 (50 μL). Asdescribed in FIG. 5 anagen phase in 5 week old mice wild type mice(C57BL/6) was induced by depilation of their dorsal hair using WaxStrips (Del Laboratories, Farmingdale, N.Y.) following themanufacturer's instructions. Note, the hair growth in minoxidil(rogaine) treated mice was not appreciably different to vehicle treatedmice. Controls were treated with empty liposome vehicle formulations andthe procedures for SPR4 in loaded vehicle formulations.

FIG. 7 shows data for contrast-images from FIG. 6 were digitallyquantitated using a pixel conversion program GelQuant.Net(BiochemicalLabSolutions.com). The lower the reading the greater thehair density/growth occurring. Note, mice treated with SPR4 had asignificant and major increase in hair density/growth compared to bothvehicle and minoxidil treated mice. Also, there was no significantdifference between minoxidil and control treated mice.

It was found that an overexpression of the enzyme 5-αreductase and thusincreased conversion of testosterone to 5-α-dihydrotestosterone (5α-DHT)plays a major role in the development of androgenetic alopecia (ADA). Toexploit this pathway we used B6CBAF1/J hybrid male mice reported to besusceptible to ADA. As a control and to remove background testosteroneeffects all the B6CBAF1/J hybrid male mice were castrated and split intotwo groups (n=6). The first group served as controls (Vehicle) and thesecond experimental group was treated with 5-α-dihydrotestosterone(5α-DHT). The 5α-DHT was administered by sub-dermal transplantation ofslow release pellets purchased from Innovative Research of America. Thecontrol group was also transplanted with identically formulated placebopellets (without 5α-DHT). Both pellets were transplanted below the rightear using a small incision that was sealed with staples.

Specifically, two 21 day release and one 90 day release pelletscontaining 10 mg of 5α-DHT respectively were used for each experimentalmouse with corresponding placebo pellets for the controls (n=6). Hairloss of the mice and the effects of 5α-DHT were then monitored over 5months as described below.

It was found that 5α-DHT mice have increased bone mineral density (BMD)and weight & lean mass. Using dual energy x-ray absorptiometry (DEXA) weshowed that castrated mice treated with 5α-DHT had major increases inweight and Bone Mass (Bone mineral Density [BMD] and Bone MineralContent [BMC]).

FIGS. 8A-8B includes DEXA analysis pictures of representative Vehicleand 5α-DHT male castrated mice showing increased bone and lean mass withtreated mice. The graph shows an increase in weight in mice treated with5α-DHT over the 5 months of the study.

Two-way ANOVA analysis of the temporal weight change show significantdifferences for 5α-DHT treatment (p<0.0001) and change over time(p<0.0001) with no significant interaction (n=6). This confirms 5α-DHTpellet infusion was effective (see also below). A pictorialrepresentation of the DEXA analysis with changes in weight are shown inFIG. 8B.

Detailed quantitative analyses of the DEXA experiments are presentedgraphically in FIG. 9. FIG. 9 shows castrated male mice infused with5α-DHT show increased bone mass, mineral content, lean mass and lean/fatmass ratios compared to vehicle infused castrated male mice (n=6). Totaland femur analyses are shown and the numbers shown on the histogramsabove the asterisks (*) are p values calculated using a two-tailedunpaired t test at 95% confidence intervals. A P<0.05 is consideredsignificant and NS indicates not significant.

These data confirm statistically significant changes in bone mass, leanmass and lean/fat mass ratios and show 5α-DHT infusion of castrated malemice is effective. Further high resolution confirmation of 5α-DHTinduced changes to bone were acquired by micro computed tomographic(μCT) analyses of femurs. Three dimensional illustrations of thesechanges are shown in FIG. 3. The 3D images show major increases incancellous bone and trabecular architecture at the top of the epiphyses.Thus infusion of 5α-DHT corrects the bone loss and abnormal boneoccurring in castrated male mice (FIG. 10).

FIG. 10 shows high resolution (6 uM) 3D μCT images of femurs fromvehicle and 5α-DHT treated castrated male mice. The arrows highlight themassive increase in cancellous and trabecular bone in the epiphyses ofexperimental mice.

In summary these experiments demonstrate and confirm the effectivenessof 5α-DHT infusion by sub-dermal pellet in castrated male mice.Castrated male mice infused with 5α-DHT develop androgenetic alopecia(AGA) were used in the studies. To determine whether 5α-DHT infusioninduced hair loss, mice were photographed with high resolution camerasover 5 months of treatment and compared with vehicle (placebo pellet)controls. Lighting, focal length, camera lens and camera were kept thesame. Also, mice were placed on a template and kept in the same order toensure reproducibility and photographic image consistency. Photographswere then analyzed using ADOBE Photoshop software and quantified andcompared by measuring integrated pixel density.

The regions compared and measured were of the same dimensions andencompassed identical locations on the head and dorsal region of theshoulder blades. Of note, hair from the region surrounding placebopellet implantation grew well but this contrasted dramatically with the5α-DHT implant region (see FIG. 11). FIG. 11 includes representativepictures of male castrated mice treated with vehicle or 5α-DHT. Notehair loss from head and shoulders with experimental mouse. Also, theimplant site shows major hair loss in mice treated with 5α-DHT mice butwas normal with placebo mice.

The hair loss from the head and shoulder blade region is also shown inFIG. 4 and FIG. 12 graphically and quantitatively depicts the reducedhair loss in mice infused with 5α-DHT. FIG. 12 shows accelerated hairloss occurred with male mice treated with 5α-DHT with an inflectionpoint after 2 months. Photographs were analyzed using ADOBE Photoshop.The inverse function of the integrated pixel density is shown on theX-axis (the lower the numbers the less hair). Two-way ANOVA analysis ofthe temporal inverse integrated pixel density showed significantdifferences for 5α-DHT treatment (p<0.0001) and change over time(p<0.0001) with significant interaction (n=6). This confirms 5α-DHTpellet infusion was effective at inducing hair loss.

A second method was then used to directly quantitate hair density, hairquality and vellus hair occurrence. Specifically, mice were sacrificedand the pelts removed and fixed in buffered formalin for histology.Also, a plastic template with a fixed portal was used to demarcate adefined and identical region of interest (ROI) between the ears andabove the shoulder blades (FIG. 12). Hair was then plucked from thisregion weighed and microscopically analyzed. Mice treated with 5α-DHTshowed significantly reduced hair density, size and quality compared toplacebo or control mice (FIG. 13). Detailed histological analysis ofparaffin embedded sections are ongoing and will be reported later.

FIG. 13 shows the pattern hair baldness is induced in castrated malemice treated with 5α-DHT. A plastic template with portal removed wasused to facilitate hair removal from defined region of interest (ROI).Top pictures comparing control (right) and experimental (left) hairsremoved from the ROI. Note smaller size and vellus like quality of thetreated mice hair, and the bottom graph shows hair density (mg/100 mm²)of vehicle treated and 5α-DHT treated mice. Hair density issignificantly less with the 5α-DHT treated mice.

The numbers shown on the histograms above the asterisk (*) is the pvalue calculated using a two-tailed unpaired t test at 95% confidenceintervals. A P<0.05 is considered significant.

The peptide can be encapsulated in a delivery composition, such as aliposome or micro emulsion or nano emulsion. The encapsulationefficiency can be 44.3% to 79.8% to 87.7% in the composition as perexperimental data. As such, the encapsulation efficiency can range fromabout 40% to about 90% or possibly higher. The composition can beprepared as described herein. In any event, a micro emulsion or nanoemulsion can be loaded with the peptide at these efficiencies.

FIGS. 14A-14D shows data from intensity dynamic light scattering sizemeasurement and zeta potential of a 15× dilution of four exampleformulations. Thus, the particles (e.g., liposomes) having the peptidecan have a size or size distribution as per FIGS. 14A-14D. The mediandiameter can be about 382.2 or 295.4 or 152.8 or 166.1 nm, or +/−1%, 2%,5%, 10%, or 20% thereof. The mean diameter can be as presented in thesefigures. The particle size range can also be up to about 430 nm or assmall as about 66.1 nm.

One skilled in the art will appreciate that, for this and otherprocesses and methods disclosed herein, the functions performed in theprocesses and methods may be implemented in differing order.Furthermore, the outlined steps and operations are only provided asexamples, and some of the steps and operations may be optional, combinedinto fewer steps and operations, or expanded into additional steps andoperations without detracting from the essence of the disclosedembodiments.

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended asillustrations of various aspects. Many modifications and variations canbe made without departing from its spirit and scope, as will be apparentto those skilled in the art. Functionally equivalent methods andapparatuses within the scope of the disclosure, in addition to thoseenumerated herein, will be apparent to those skilled in the art from theforegoing descriptions. Such modifications and variations are intendedto fall within the scope of the appended claims.

The present disclosure is to be limited only by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled. It is to be understood that this disclosure is notlimited to particular methods, reagents, compounds compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of“A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” and the like include the number recited andrefer to ranges which can be subsequently broken down into subranges asdiscussed above. Finally, as will be understood by one skilled in theart, a range includes each individual member. Thus, for example, a grouphaving 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, agroup having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells,and so forth.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims.

All references recited herein are incorporated herein by specificreference in their entirety: U.S. Ser. No. 11/521,684; and U.S. Pat. No.7,825,217.

REFERENCES

-   David V, Martin A C, Hedge A M, Drezner M K, Rowe P S. ASARM    peptides: PHEX-dependent & independent regulation of serum    phosphate. Am J Physiol Renal Physiol. 2011; 300(3):F783-91.-   Atkins G J, Rowe P S, Lim H P, Welldon K J, Ormsby R, Wijenayaka A    R, Zelenchuk L, Evdokiou A, Findlay D M. Sclerostin is a locally    acting regulator of late-osteoblast/pre-osteocyte differentiation    and regulates mineralization through a MEPE-ASARM dependent    mechanism. J Bone Miner Res. 2011; 26(7): 1425-36.-   Martin A, David V, Laurence J S, Schwarz P M, Lafer E M, Hedge A M,    Rowe P S.-   Degradation of MEPE, DMP1, and release of SIBLING ASARM-peptides    (minhibins): ASARM-peptide(s) are directly responsible for defective    mineralization in HYP. Endocrinology. 2008; 149(4): 1757-72.-   David V, Martin A, Hedge A M, Rowe P S. Matrix extracellular    phosphoglycoprotein (MEPE) is a new bone renal hormone and    vascularization modulator. Endocrinology. 2009; 150(9):4012-23.-   Rowe P S, Matsumoto N, Jo O D, Shih R N, Oconnor J, Roudier M P,    Bain S, Liu S, Harrison J, Yanagawa N. Correction of the    mineralization defect in hyp mice treated with protease inhibitors    CA074 and pepstatin. Bone. 2006; 39(4):773-86.-   Rowe P S N, Garrett I R, Schwarz P M, Carnes D L, Lafer E M, Mundy G    R, Gutierrez G E. Surface Plasmon Resonance (SPR) confirms MEPE    binds to PHEX via the MEPE-ASARM-motif: A model for impaired    mineralization in X-linked rickets (HYP). Bone. 2005; 36(1):33-46.-   Rowe P S, Kumagai Y, Gutierrez G, Garrett I R, Blacher R, Rosen D,    Cundy J, Navvab S, Chen D, Drezner M K, Quarles L D, Mundy G R. MEPE    has the properties of an osteoblastic phosphatonin and minhibin.    Bone. 2004; 34(2):303-19. PMCID: 3357088.-   Yuan B, Takaiwa M, Clemens T L, Feng J Q, Kumar R, Rowe P S, Xie Y,    Drezner M K. Aberrant Phex function in osteoblasts and osteocytes    alone underlies murine X-linked hypophosphatemia. J Clin Invest.    2008; 118(2):722-34.-   Bresler D, Bruder J, Mohnike K L, Fraser D, Rowe P S N. Serum    MEPE-ASARM-peptides are elevated in X-linked rickets (HYP):    implications for phosphaturia and rickets. J Endocrinol. 2004;    183:R1-9.-   Pfaffl M W. A new mathematical model for relative quantification in    real-time RT-PCR. Nucleic Acids Res. 2001; 29(9):e45. PMCID: 55695.-   Gluhak-Heinrich J, Ye L, Bonewald L F, Feng J Q, MacDougall M,    Harris S E, Pavlin D. Mechanical loading stimulates dentin matrix    protein 1 (DMP1) expression in osteocytes in vivo. J Bone Miner Res.    2003; 18(5):807-17.-   Verma D D, Fahr A. Synergistic penetration enhancement effect of    ethanol and phospholipids on the topical delivery of cyclosporin A.    Journal of controlled release: official journal of the Controlled    Release Society. 2004; 97(1):55-66.-   Crabtree J S, Kilbourne E J, Peano B J, Chippari S, Kenney T,    McNally C, Wang W, Harris H A, Winneker R C, Nagpal S, Thompson C C    2010 A mouse model of androgenetic alopecia. Endocrinology    151(5):2373-80.-   Park H J, Zhang N, Park D K 2011 Topical application of Polygonum    multiflorum extract induces hair growth of resting hair follicles    through upregulating Shh and beta-catenin expression in C57BL/6    mice. J Ethnopharmacol 135(2):369-75.-   Park W S, Lee C H, Lee B G,Chang I S 2003 The extract of Thujae    occidentalis semen inhibited 5alphareductase and androchronogenetic    alopecia of B6CBAF1/j hybrid mouse. J Dermatol Sci 31(2):91-8.-   Matias J R, Malloy V, Orentreich N 1989 Animal models of    androgen-dependent disorders of the pilosebaceous apparatus. 1. The    androchronogenetic alopecia (AGA) mouse as a model for malepattern    baldness. Arch Dermatol Res 281(4):247-53.-   Matias J R, Orentreich N 1988 The effect of testosterone,    cyproterone acetate, and minoxidil on hair loss in the    androchronogenetic alopecia mouse. Clin Dermatol 6(4):169-76.

1. A topical composition comprising: a pharmaceutical carrier configuredfor topical application to a subject; and a polypeptide in thepharmaceutical carrier and having a sequence that has at least 75%complementarity to or at least 75% identical to SPR4, wherein SPR4 is:TVNAFYSASTNYPRSLSYGAIGVIVGHEFTHGFDNNGRGENIADNG (SEQ ID NO: 1).
 2. Thetopical composition of claim 1, wherein the polypeptide has at least 80%complementarity to or is at least 80% identical to SPR4.
 3. (canceled)4. (canceled)
 5. The topical composition of claim 1, wherein thepolypeptide has at least 99% complementarity to or is at least 99%identical to SPR4.
 6. The topical composition of claim 1, wherein thepharmaceutical carrier is a liposome or micro emulsion or nano emulsion.7. The topical composition of claim 1, wherein the polypeptide isincluded in a fusion polypeptide with a second polypeptide, wherein thesecond polypeptide includes an endosomal disrupting polypeptide. 8.(canceled)
 9. (canceled)
 10. The topical composition of claim 1, whereinthe polypeptide is present in an amount sufficient to: increase hairgrowth in a subject upon topical application to the subject; or increasehair follicle growth/development in a subject upon topical applicationto the subject.
 11. (canceled)
 12. (canceled)
 13. The topicalcomposition of claim 1, wherein the carrier includes one or more ofcetearyl alcohol, cetearyl glucoside, squalane, isopropyl palmate,octyldodecaonol, phenoxyethanol, methylparaben, etheylparaben,butylparaben, propylparaben, isobutylparaben, glycerin, butylene glycol,sodium acrylate, acryloyldimethyl taurate, isohexadecane, polysorbate,glyceryl stearate, dicaprylyl ether, alkyl benzoate, isononylisononanoate, methylpropanediol, tetrasodium EDTA, iodoproynylbutylcarbamate, triethanolamine, ketoconazole, serenoa serrulataextract, emu oil, niacin vitamin B3, caffeine, pyridoxine, L-pathenol,linolenic acid, simmondsia chinesis seed oil, zinc oxide, lecithin,ZnCl₂, L-α-phosphatidylcholine, ethanol, PBS, phospholipids, fattyacids, tocopherol, and derivatives thereof and equivalents thereof. 14.The topical composition of claim 1, further comprising an active hairgrowth agent.
 15. The topical composition of claim 14, wherein theactive hair growth agent is selected from minoxidil and finasteride. 16.(canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. The topicalcomposition of claim 1, wherein the polypeptide is contained in aliposome or microsphere or polymer particle or lipid emulsion orcombination thereof.
 21. A method of promoting hair growth, the methodcomprising: providing a polypeptide having a sequence that has at least75% complementarity to or at least 75% identical to SPR4, wherein SPR4is: TVNAFYSASTNYPRSLSYGAIGVIVGHEFTHGFDNNGRGENIADNG (SEQ ID NO: 1); andtopically administering the polypeptide to a subject.
 22. The method ofclaim 21, comprising providing a composition having a pharmaceuticalcarrier configured for topical application to a subject having thepolypeptide; and administering the composition topically so as toadminister the polypeptide to the subject.
 23. The method of claim 21,comprising administering the polypeptide to: skin of the subject; a hairfollicle of the subject; or a bald spot of the subject.
 24. (canceled)25. (canceled)
 26. The method of claim 21, comprising administering thepolypeptide so as to: modulate the Wnt/beta-catenin canonical pathway;regulate one or more genes involved in hair growth; accelerate hairgrowth; lengthen the anagen phase; reduce 5-alpha-reductase; suppresssclerostin; and/or increase active beta-catenin.
 27. (canceled) 28.(canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled)33. (canceled)
 34. The method of claim 21, comprising administering thepolypeptide in an amount to: treat alopecia and/or related syndromes;lengthen the anagen phase; increase hair growth on a bald spot; and/orincrease hair follicle growth.
 35. (canceled)
 36. (canceled) 37.(canceled)
 38. The method of claim 21, comprising administering thepolypeptide in an amount to increase hair growth compared to when thepolypeptide is not administered.
 39. The method of claim 21, comprisingadministering the polypeptide by micro-needle injection.
 40. The methodof claim 39, wherein the micro-needle injection is performed by aderma-roller.
 41. The method of claim 39, wherein the skin is notvisibly damaged by the micro-needle injection.
 42. The method of claim39, comprising forming micro-pores in the skin with the micro-needle,and allowing the micro-pores to close after the micro-needle injection.